Microarrays of biomolecules, such as DNA, RNA, cDNA, polynucleotides, oligonucleotides, proteins, and the like, are state-of-the-art biological tools used in the investigation and evaluation of biological processes, including gene expression and mutation for analytical, diagnostic, and therapeutic purposes. Microarrays typically comprise a plurality of polymers, e.g., oligomers, synthesized or deposited on a substrate in an array pattern of features. The support-bound polymers are typically called “probes”, which function to bind or hybridize with a sample of polymer material under test, i.e., a moiety in a mobile phase (typically fluid), called a “target” in hybridization experiments. However, some investigators also use the reverse definitions, referring to the surface-bound polymers as targets and the solution sample of polymer as probes. Further, some investigators bind the target sample under test to the microarray substrate and put the polymer probes in solution for hybridization. Either of the “target” or “probes” may be the one that is to be evaluated by the other (thus, either one could be an unknown mixture of polymers to be evaluated by binding with the other). All of these iterations are within the scope of this discussion herein. The plurality of probes and/or targets in each location in the array is known in the art as a “feature”. A feature is defined as a locus onto which a large number of probes and/or targets all having the same monomer sequence are immobilized. In use, the array surface is contacted with one or more targets under conditions that promote specific, high-affinity binding of the target to one or more of the probes. The targets are typically labeled with an optically detectable label, such as a fluorescent tag, dye or fluorophore, so that the targets are detectable with scanning equipment after a hybridization assay. DNA array technology, for example, offers the potential of using a multitude (hundreds of thousands) of different oligonucleotides to analyze changing mRNA populations.
Typical scanning equipment used for biomolecular analysis, such as scanning fluorometers, comprise an excitation light source, an optical system for directing light to and from a sample being scanned, a detection system and optionally an analysis system. To analyze a microchip after a hybridization assay, the scanner scans a light from its excitation light source across the microchip. The light excites the optically detectable labels on the hybridized biomolecules. The excited labels in turn emit light at one or more particular wavelengths. The emitted light from the biomolecules is detected and measured by the detection system and the measurements are analyzed by analysis equipment to determine the results of the assay. In competitive hybridization assays, more than one label may be used, each of which emit light having a characteristic emission spectrum, which may be narrow or broad, to distinguish the biomolecules on the microchip. The light emitted by each different label must be separately detected by the scanning equipment for analysis. State-of-the-art scanners are equipped with a detection system having multiple channels for detecting emissions at different wavelengths, for example. The detection systems having multiple channels are designed to detect signals from a combination of dyes or dyes having broad emission spectra that are used in labeling. Parameters such as the intensity, the wavelength, and the location of the emitted light on the microchip provide important information about the target material being assayed. Therefore, accurate measurement of these parameters is essential to providing accurate information about the target material.
The detection systems used in scanning equipment comprise one or more detector components, such as photomultiplier tubes (PMTs). PMTs are known to age and to also deteriorate as a function of signals and overloads previously received.
An approach to improving the accuracy of a fluorometer used for scanning flow cells is to determine the relative fluorescence intensity or index (RFI) for a bulk sample in the flow cell. Gifford et al., U. S. Pat. Nos. 4,750,837 and 4,802,768, discuss and illustrate approaches for compensating for variations in excitation light using reference signals or reference paths, and the advantages and disadvantages of these approaches, and further, disclose a reference system that accounts for variations in signal levels from light sources and detection components that affect the RFI. A computation is made on measurements taken on the detection components and light sources, which indicates the relative concentration of the target being assayed using the flow cell. The computation is intended to cancel out the variations in the light sources and the detectors. The system described by Gifford et al. provides only a relative measurement of the target concentration in a bulk sample using flow cells. Therefore, there is little or no consistency between fluorometer systems taught by Gifford et al.
Thus, it would be advantageous to have a scanning system for scanning microarrays of biomolecules on microchips that is self-calibrating in that the sensitivity changes in the detection components are compensated for. Further, it would be advantageous to have a scanning system that could provide absolute target concentration measurement results where the results are reproducible from scanner to scanner. Still further, it would be advantageous if such self-calibration could be integrated into the scanner and the calibration be performed automatically.